In Vitro Activity of Terbinafine Combined with Caspofungin and Azoles against Pythium insidiosum

Abstract

In this text we evaluated the in vitro antifungal activities of terbinafine combined with caspofungin, miconazole, ketoconazole, and fluconazole against 17 Pythium insidiosum strains by using the microdilution checkerboard method. Synergistic interactions were observed with terbinafine combined with caspofungin (41.2% of the strains), fluconazole (41.2%), ketoconazole (29.4%), and miconazole (11.8%). No antagonistic effects were observed. The combination of terbinafine plus caspofungin or terbinafine plus fluconazole may have significant therapeutic potential for treatment of pythiosis.

Pythiosis is a life-threatening infectious disease in humans and animals that is caused by the aquatic oomycete Pythium insidiosum (9). Horses are the most frequently infected animals, and equine pythiosis typically involves ulcerative granulomas (8). In humans, the infection occurs as ophthalmic, subcutaneous, and systemic forms, which are frequently associated with α- and β-thalassemia (5, 7). Pythiosis therapy, which is based on amphotericin B or azoles, has been ineffective or controversial, and the associated prognosis for human and equine pythiosis is poor (5, 7, 8, 9, 12). Therefore, surgical procedures, including amputation, are often effective, but disease reoccurrence rates are unfortunately high (7).

Combinations of antifungal agents against pythiosis have not been thoroughly studied, and therefore, such in vitro combinatory activities against P. insidiosum require attention (1, 6).

The purpose of this study was to investigate the in vitro activity of terbinafine (TRB) combined with caspofungin (CAS), miconazole (MNZ), ketoconazole, and fluconazole (FLC) against 17 strains of Pythium insidiosum isolated from animals.

A total of 15 Brazilian P. insidiosum strains isolated from equines with pythiosis and two standard strains (ATCC 58637 and CBS 101555) were tested. All strains were maintained in cornmeal agar, and strain identification was confirmed by a PCR-based assay (4).

The susceptibility of the P. insidiosum strains to the antifungal agents was tested by microdilution, based on protocol M38-A2 (2). The inoculum consisted of P. insidiosum zoospores obtained following zoosporogenesis. Cell numbers of zoospores were counted on a hemocytometer; zoospores were diluted in RPMI 1640 containing l-glutamine and buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid) to obtain a final concentration range of 2 × 10³ to 3 × 10³ zoospores/ml (10).

The combinations of TRB (Novartis) plus CAS (Merck), TRB plus MNZ (Labware), TRB plus ketoconazole (Janssen), and TRB plus FLC (Pfizer) were evaluated using the checkerboard technique, according to the broth microdilution design (2, 14). In the individual tests, 100 μl of each drug concentration was plated in microplate wells and an equal volume of the inoculum was added to each well. In the combination tests, the antifungals were plated at a 4× concentrate of 50 μl of drug A plus 50 μl of drug B and 100 μl of the inoculum, resulting in a final 1× drug concentration of each compound. The microplates were incubated at 37°C for 24 h. The MIC was defined as the lowest drug concentration at which there was 100% inhibition of fungal growth by visual readings. The tests were performed in duplicate, and the assay was repeated when disparate values were obtained. The interactions, based on the respective fractional inhibitory concentration index (FICI), were interpreted as the following: FICI ≤ 0.5, synergism; FICI > 0.5 to ≤4, indifference; FICI > 4, antagonism. FICIs were obtained using the formula FICI = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone).

The in vitro activities of individual antifungal agents against P. insidiosum are shown in Table 1. In general, the patterns of susceptibility demonstrated that individual drugs had only weak antifungal activity or none.

TABLE 1.

In vitro activities of TRB, CAS, and azoles against isolates of Pythium insidiosum

Drug	MIC (mg/liter) (n = 17)
	Rangea	GMb	MIC50	MIC90c
TRB	8-32	14.7	16	32
CAS	8-64	19.6	16	64
MNZ	4-32	13.6	16	32
Ketoconazole	16-64	23.1	32	64
FLC	32-64	59.0	64	64

^aRange between the lowest and highest MICs for all isolates.

^bGM, geometric mean of MIC.

^cMIC of drug capable of inhibiting the growth of 90% of isolates.

The combinations of TRB plus FLC and TRB plus CAS both exhibited synergistic effects against seven (41.2%) P. insidiosum strains. The combination of TRB plus ketoconazole was also synergistic against five (29.4%) isolates, while the combination of TRB plus MNZ exhibited synergistic effects against only two (11.8%) isolates (Table 2). Antagonistic effects were not indicated.

TABLE 2.

In vitro activity of TRB combined with FLC, MNZ, ketoconazole, or CAS against Pythium insidiosum^c

Isolatea	TRB and FLC			TRB and MNZ			TRB and ketoconazole			TRB and CAS
	MIC of combination (mg/liter)		FICI (interpret.)b	MIC of combination (mg/liter)		FICI (interpret.)	MIC of combination (mg/liter)		FICI (interpret.)	MIC of combination (mg/liter)		FICI (interpret.)
	TRB	FLC		TRB	MNZ		TRB	Ketoconazole		TRB	CAS
LAPEMI 119	4	0.125	0.2 (S)	4	0.5	0.3 (S)	4	0.125	0.3 (S)	0.5	1	0.1 (S)
LAPEMI 123	8	0.125	1.0 (I)	0.25	16	0.6 (I)	0.25	16	1.0 (I)	0.25	8	0.6 (I)
LAPEMI 124	32	64	2.0 (I)	1	16	1.0 (I)	0.25	1	0.5 (S)	0.25	16	0.5 (S)
LAPEMI 125	32	64	2.0 (I)	32	32	2.0 (I)	32	32	2.0 (I)	4	16	0.6 (I)
LAPEMI 126	4	0.125	0.5 (S)	0.25	16	1.0 (I)	4	8	0.6 (I)	0.25	8	0.6 (I)
LAPEMI 128	16	0.125	1.0 (I)	0.25	16	1.0 (I)	0.25	8	1.0 (I)	0.25	32	0.2 (S)
LAPEMI 129	8	0.125	1.0 (I)	1	32	1.1 (I)	4	8	0.6 (I)	0.25	8	0.6 (I)
LAPEMI 130	8	4	0.3 (S)	0.25	16	2.0 (I)	0.25	4	0.5 (S)	0.25	8	0.5 (S)
LAPEMI 135	8	0.125	0.5 (S)	0.25	0.25	0.1 (S)	0.25	16	0.6 (I)	0.25	8	0.6 (I)
LAPEMI 136	8	4	0.6 (I)	0.25	16	1.0 (I)	0.25	0.5	0.1 (S)	0.25	4	0.2 (S)
LAPEMI 138	32	0.125	2.0 (I)	4	8	1.2 (I)	8	4	0.6 (I)	0.25	16	0.6 (I)
LAPEMI 144	4	0.125	0.5 (S)	0.25	16	1.0 (I)	4	8	0.6 (I)	1	4	1.1 (I)
LAPEMI 147	32	0.125	2.0 (I)	16	0.25	1.0 (I)	32	0.125	2.0 (I)	0.25	16	1.0 (I)
LAPEMI 148	16	16	2.0 (I)	0.25	8	0.6 (I)	0.25	16	1.0 (I)	0.25	0.5	0.1 (S)
LAPEMI 187	4	0.125	0.5 (S)	4	0.25	0.6 (I)	4	0.125	0.5 (S)	4	0.5	0.5 (S)
ATCC 58637	8	8	0.7 (I)	0.25	8	1.0 (I)	0.25	16	1.0 (I)	0.25	4	1.0 (I)
CBS 101555	8	0.125	0.5 (S)	0.25	8	2.0 (I)	4	16	0.7 (I)	1	8	0.6 (I)

^aLAPEMI, Laboratório de Pesquisas Micológicas; ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures.

^binterpret., interpretation; S, synergistic; I, indifferent.

^cn = 17.

The use of combination therapy in the treatment of pythiosis could be an alternative to monotherapy (3), but such application would require further investigation. Herein, we examined the in vitro activities of selected antifungal agents singularly or in combination against P. insidiosum.

Our results are difficult to interpret, since only a few previous studies investigating the susceptibility of P. insidiosum have been reported, and those studies were performed using different experimental techniques (1, 13). In addition, the breakpoints for susceptibility tests with antifungal agents against P. insidiosum are not defined (2). Therefore, these results suggest relatively weak antifungal activity of the individual agents, which is in accordance with the well-known therapeutic failures in pythiosis treatment. In contrast, the results obtained utilizing drug combinations, which are based on FICIs, can be interpreted with more confidence for activity.

Studies focusing on the use of combination therapy against P. insidiosum are almost nonexistent. The first report on the potential synergistic effects of TRB plus itraconazole was that by Shenep et al. (13), which involved the successful treatment of deeply invasive facial infections from P. insidiosum infection in a child with this combination therapy.

Argenta et al. (1) reported that the combination of TRB and either itraconazole or voriconazole was synergistic against 17% of the strains tested and no antagonistic effects were observed. In this study, we demonstrated significant synergistic effects, as the combinations of TRB plus FLC and TRB plus CAS were synergistic against 41.2% of P. insidiosum strains. Additionally, synergistic effects were indicated with combinations of TRB plus ketoconazole and TRB plus MNZ, albeit to a lower extent (against 29.4% and 11.8% of strains, respectively). To our knowledge, the synergistic effects of these antifungals against P. insidiosum are being reported for the first time in this study, and antagonistic effects of these combination antifungal treatments were not observed.

However, a concern for the use of combination antifungal therapy in treating P. insidiosum infection is the great variation in susceptibility among the different strains, which may be related to the genetic variability of the strains tested (11). However, our in vitro results demonstrate that combination antifungal therapy may be an alternative in the treatment of P. insidiosum. In vivo studies must be further investigated experimentally, since 48.5% of the combined MICs were lower than the serum concentrations achieved by the respective agents, which indicates the potential therapeutic utility of our results.